Substrate amounts of rat brain cerebellar nitric oxide synthase (NOS) in the absence of NADPH or any other added electron donor convert arginine predominantly to N-hydroxyarginine (NOHA). Conversion of arginine to NOHA requires calmodulin, and is stimulated 3-fold by tetrahydrobiopterin (BH4). Upon addition of NADPH, arginine is converted exclusively to citrulline. The conversion of NOHA to citrulline requires NADPH, and, like the first partial reaction, also requires calmodulin and is stimulated 3-fold by BH4. BH4 could not substitute for NADPH. These combined results indicate that the endogenous electron donor for the first partial reaction is not active in the second (NADPH-dependent) partial reaction. NOS contains bound BH4 and a flavin semiquinone radical, each of which are potential electron donors. We now have tentative evidence that NOS also contains hound NADPH (1 mole/mole NOS subunit). The role of each of these electron donors in the first partial reaction is currently being examined. In order to assess the role of BH4 in NOS, we have attempted to express a rat brain NOS clone in an organism such as E.coli that cannot synthesize BH4. Protein of the expected size (150 kD subunits) was expressed, but accumulated in insoluble and inactive forms. We have also expressed the cytochrome P450 reductase (CPR)-like domain by fusion to maltose binding protein (MBP). The fused protein was purified to near homogeneity by amylose affinity chromatography, and its properties were compared to those of native NOS. Experiments are underway to express the oxygenase domain of brain NOS with the goal of complementing the CPR domain and thereby reconstructing complete NOS activity.